Novel recognition and targeting of temozolomide resistant cells in glioblastoma Project Summary Glioblastoma (GBM) is the most common and deadly primary brain tumor with an incidence of approximately 14,000 new cases annually in the United States. Despite surgery and treatment with radiation and temozolomide (TMZ), median survival is only about 18 months. Once GBM recurs, it is universally fatal with survival of less than a few months despite aggressive treatment. Resistance to TMZ, the main chemotherapeutic for GBM, is a major problem. Resistance can occur through selection of TMZ-resistant cells in the tumor as well as conversion of cells to a resistant state. Importantly, rapid identification and enrichment of TMZ-resistant cells from resected patient tumors could lead to new and innovative treatments targeting resistant cells. We found that TMZ-resistant glioma cell lines significantly differ from parent cells in the whole cell electrophysiological property membrane capacitance. We propose to enrich low capacitanceTMZ-resistant cells using an innovative platform we developed to sort cells on the basis of whole cell membrane capacitance. We found cells differing in membrane capacitance also vary in cell surface glycosylation, providing a molecular basis for capacitance differences between cells. We will test whether sorting cells on the basis of N-glycans enables isolation of TMZ-resistant cells. Glycosylation impacts function of other cancer cell types, leading to changes in tumor growth, migration and invasion, but whether glycosylation plays a key role in resistance of GBM cells is not known. Therefore, we will also test whether changing cell surface glycosylation alters susceptibility of cells to TMZ. We hypothesize that TMZ-resistant cells in patient GBM tumors will (a) have distinct membrane capacitance values enabling their isolation and (b) express different patterns of cell surface glycosylation compared to non-resistant cells, which can impact cell function and potentially sensitivity to chemotherapeutics. In Aim 1 we will test whether patient-derived TMZ-resistant cells can be enriched by membrane capacitance. In Aim 2 we will test whether distinct cell surface N-glycans can be used to enrich TMZ-resistant cells and whether altering N-glycans impacts drug sensitivity. A means to rapidly isolate TMZ-resistant cells from tumor samples would enable their molecular characterization and identify novel treatment strategies. Tying membrane capacitance of resistant cells to cell surface glycosylation could identify novel therapeutic strategies, including treatment of patients with well-tolerated pharmacological agents affecting glycosylation that render cells sensitive to chemotherapeutics.